Semaphorin4D and PlexinB1 mediate GABAergic synapse development in mammalian CNS. Proper neuronal communication depends on the precise assembly and development of synaptic connections between neurons. Many neurological disorders arise from perturbations in synaptic connectivity, such as mental retardation, autism, and Rett Syndrome (Fernandez and Garner, 2007; Rubenstein and Marzenich, 2003; Tabuchi et al., 2007; Zoghbi, 2003). In addition, many synaptic proteins, including Semaphorin family members, have been linked to neurodevelopmental disorders (Weiss et al, 2009) and the lack of Semaphorins results in improper synaptic connectivity in rodents (Morita et al., 2006; O'Connor et al., 2009; Paradis et al., 2007; Sahay et al., 2005). The transmembrane protein Sema4D is necessary for proper GABAergic synapse formation, as knockdown of expression in the postsynaptic neuron by RNAi leads to a decrease in GABAergic synaptic density in cultured neurons (Paradis et al 2007). Our preliminary results demonstrate that addition of the soluble, extracellular domain of Sema4D to cultured hippocampal neurons is sufficient to drive GABAergic synapse formation. Importantly, this increase is dependent on the expression of Sema4D's receptor, PlexinB1. In addition, our voltage-clamp experiments indicate that PlexinB1 is necessary for proper GABAergic synaptic transmission in the hippocampus. Thus, our work defines PlexinB1 as a novel receptor mediating GABAergic synapse formation in response to Sema4D in the mammalian CNS. The goal of this proposal is to elucidate the role of Sema4D and it's receptor, PlexinB1 in GABAergic synapse development. In experiments proposed here, we hypothesize that Sema4D acts to initiate assembly of GABAergic synaptic proteins such as GABAA receptors and gephyrin through its receptor PlexinB1. This will be tested using a variety of imaging techniques in cultured hippocampal neurons, including confocal and time- lapse imaging, to measure the mobility and accumulation of GABAergic synaptic proteins in neurons after treatment with soluble Sema4D. In addition, preliminary data suggests that PlexinB1 is important for GABAergic synaptic transmission. Electrophysiology will be used to determine the site of action of PlexinB1 function to regulate GABAergic synaptic transmission. The experiments proposed here will not only greatly expand our understanding of a novel receptor-ligand pair in GABAergic synapse development; it will inform us as to some of the basic mechanisms underlying GABAergic synaptogenesis. ! PUBLIC HEALTH RELEVANCE: Research of neurodevelopmental disorders such as Autism Spectrum Disorders (ASDs) has demonstrated that it is vital to establish correct neuronal connections for proper cognitive function. The importance of proteins that regulate synapse development such as Semaphorin family members in neurological disorders has come to light recently through linkage analyses of families with ASDs. In this proposal, I wish to explore the role of Semaphorin4D and it's receptor, PlexinB1, in regulating inhibitory synapse development and ultimately setting up proper neural connections in the mammalian CNS.